A couple of possible causes:
1 - Too much air in the solution
2 - TEMED too old
3 - APS not fresh
I use water for overlayering the gel and minigels polymerize in 35-40
minutes with 5 µl TEMED so I do not think that the water is a problem. If
your acrylamide is reacently made up there might be a lot of oxygen in it,
and I would highly recommend degassing. Did you try to make the buffers
over again? Things can always happen. I hope this can help so GOOD LUCK !
> Recently, when I adapted my old protocal for a mini-gel, it takes hours for the separating gel to solidify and even worse, the stacking gel solidified poorly and the wells did not contain the samples very well as the result of it.
>> The way I prepare the separating gel is that I don't deareate it. After
> I pour a gel into the assembly I add in some isobutanol right away. I
> found after the gel solidified, there is some water in between the
> isobutanol and the gel.
>> It takes less time now (1 hr) for the separating gel to solidify after I
> increased TEMED from 5 ul/10ml to 20 ul/10ml (10% Ammonium persulfate
> unchanged: 50 ul/10ml).